This application claims priority to PCT Application No. PCT/SE98/00891, filed May 14, 1998, and Swedish Application No. SE 9701872-5, filed May 16, 1997.
Oral implants are made of syntetic materials and inserted in mucosal soft tissues and bone to serve as anchorage for prosthetic constructions. The choice of materials for bone anchorage has been discussed and considered over the years (reviewed in (Br{dot over (a)}nemark, 1996)) Osseointegrated titanium implants ad modum Br{dot over (a)}nemark have been successfully used for 30 years. There are several factors which are assumed to play important roles for the outcome of this treatment, for instance the choice of titanium with its adequate mechanical properties and corrosion resistance (Williams, 1981), surface topography and the relatively non-traumatic surgical procedures.
It is assumed that two-stage surgical procedure with an early post-operative period (3-6 months) without loading is important for the initial implant stability during the early healing phase. However, the two-stage surgical technique may be a disadvantage for the patient and requires more resources. In clinical practice, not only the materials and surgical procedures but also systemic and local host factors set the limits for treatment. It has been found that the failure rates are higher in the maxilla and the posterior mandible and that the success rates very much depend on the quality of bone (Esposito et al., 1997). It is therefore of importance to identify the beneficial and negative factors related both to the implant and the host in order to optimize the implant treatment. A reduction of the healing period and a maintenance of long-term stability during clinical loading conditions therefore appears essential.
A biomaterial is a material used in a medical device, intended to interact with biological systems (Black, 1992). The materials used in man-made structures may be divided into three classes: metals, ceramics and polymers. The classes are distinguished by the type of interatomic bonding (Cooke et al., 1996).
Metals consist of a large number of small crystallites. Each crystallite is an aggregate of atoms regularly arranged in a crystalline structure. When molten metals (which are amorphous) solidify small crystals (grains) start to grow. The irregularly arranged crystals eventually meet each other which gives rise to boundaries between the crystals, grain boundaries. The imperfect packing of atoms in the boundaries constitutes weak points in the material, which will be most strongly affected by a surface treatment such as etching or plasma cleaning and a groove will be created showing up as a darker line. The surface properties of a material is different from the bulk properties.
The term commercially pure (CP) titanium is applied to unalloyed titanium and includes several grades containing minor amounts of impurity elements, such as carbon, iron and oxygen. The amount of oxygen can be controlled at different levels to provide increased strength. There are four grades of titanium where grade 1 (used in the present thesis) contains the lowest amount of oxygen. The microstructure of CP titanium is essentially all xcex1 titanium which has a HCP crystal structure.
Titanium dioxide, TiO2, is the most common and stable of the titanium oxides, while Ti2O3 and TiO are more rare (Lausmaa, 1991).
TiO2 can exist in three crystalline modifications; anatase (tetragonal structure), rutile (tetragonal), and brookite (orthotrombic). Rutile and anatase are the most usual forms whereas brookite is very rare (Keesman, 1966).
Techniques have been developed to alter and modify the surface properties of implants via mechanical and chemical procedures (Lausmaa, 1996; Smith et al., 1991a; Smith et al., 1991b). Plasma-spraying, sputter deposition, oxidation, vaporization, (grit, sand) blasting, grinding, etching, plasma cleaning and ion bombardment are examples of techniques available for this purpose.
Electropolishing is an electrochemical technique often used to obtain an improved surface finish by controlled dissolution of the surface layer of the metal. The amorphous surface layer produced by the machining of the implants is removed. After electropolishing a polycrystalline surface with a surface oxide consisting mainly of TiO2, typically 3-5 nm thick as measured by X-ray photoelectron spectroscopy (XPS), is found on the surface (Lausmaa, 1996).
Anodic oxidation is an electrochemical method used to increase the thickness of the oxide layer on metal implants. A current is applied in an electrolytic cell in which the sample is the anode. When a potential is applied on the sample, the current will transport oxygen containing anions through the electrolyte and a continuous oxide is formed on the metal sample. The stoichiometry of anodic oxides on titanium is mostly TiO2. The anodic oxides on titanium contain various structural features such as porosity (Lausmaa, 1996).
In order to characterize the surface properties after the modifications the following techniques were used; SEM and AFM for surface topography and roughness; ESCA and AES for surface composition and oxide thickness.
Interactions Between Titanium Surfaces and Proteins/Cells/Tissues
A review of the literature shows that surface modifications influence the biological response. The first events that take place when an implant is inserted in vivo is the exposure of the material surface to water and biomolecules, including plasma proteins. Both under in vitro and in vivo conditions serum proteins are known to adsorb to foreign material surfaces within seconds. The adsorption and desorption phenomena on different biomaterial surfaces have been studied intensely. A working hypothesis is that the biological response is directed by the initial protein adsorption which subsequently influence the cellular/tissue response and ultimately the performance of the implant (Horbett, 1996).
Three types of adsorption/desorption patterns have been described for metals and their oxides (Williams and Williams, 1988). For example, titanium was found to adsorb low levels of albumin, which remained low during a 48 h period. In addition, the albumin desorbed relatively easily. Other metal surfaces such as vanadium, showed an initially low amount of albumin, but the amount increased and desorption was slow. Gold was found to be characteristic for a surface with a high initial adsorption of albumin and the amount increased throughout the experiment.
A modification and variation on surface properties and the resulting effects on molecular adsorption to surfaces may provide important insights into the role of surface properties for biological reactions. Modified and characterized surfaces have been used to detect differences in the behaviour and adsorption patterns of proteins (McAlarney et al., 1991; McAlarney et al., 1996; Nygren, 1996; Shelton et al., 1988; Sunny and Sharma, 1990; Tengvall et al., 1992; Wxc3xa4livaara et al., 1994; Wxc3xa4livaara et al., 1992). Shelton et al (1988) found that a larger amount of proteins were adsorbed to negatively charged polymer beads than to positively charged beads but the roughness of the surface did not seem to influence protein adsorption or cellular behaviour. In general, rough surfaces are considered more wettable than smooth surfaces which may be an effect caused by an increase of the surface area as well as by an increased hydrophilicity of the surface (Curtis et al., 1983).
Nygren (1996) found two different reactions when hydrophilic and hydrophobic titanium surfaces were exposed to whole blood. On the hydrophobic surface, adherent platelets and fibrinogen were present while complement factor 1 (C1) and prothrombin/thrombin were present on the hydrophilic surface. Baier et al. (1982) has reviewed the principles of adhesive phenomena in diverse systems and he pointed out the wettability of a surface as the important parameter influencing the protein adsorption pattern.
The surface energy of a material is influenced by various cleaning procedures and the oxide thickness. According to Sunny and Sharma (1990) an increase of the oxide layer on aluminium, increased the hydrophobicity of the surface, resulting in an increased adsorption of fibrinogen. In addition, the glow discharge technique rendered the surface more hydrophilic causing less fibrinogen adsorption. However, other results were obtained by Wxc3xa4livaara et al (1994) who found that the titanium oxide thickness and carbon contamination had no influence on protein adsorption and contact activation. Interestingly, increased surface concentrations of complement factor 3 (C3) was correlated with an increasing titanium dioxide film thickness and/or crystallinity. The oxide crystallinity seemed to be of more significance than the oxide thickness (McAlarney et al., 1996). In another study, McAlarney (1991) found that C3 adsorbed preferentially onto grain boundaries which may be explained by the differences in surface energy between grain boundaries and bulk surface. It is known that titanium oxide surfaces bind cations, particulary polyvalent cations (Abe, 1982).
The oxide layer is highly polar and attracts water and water-soluble molecules. In general therefore, calcium ions may be attracted to the oxide surface by electrostatic interaction with oxygen (Oxe2x88x92). In a study by Lausmaa et al (1988), approximately 100 samples prepared according to clinical procedures were analyzed with ESCA. The spectra showed that the surface consisted mainly of TiO2. Carbon and smaller amounts of N, Cl, Ca, S, P, Na and Si were found on the surface but after sputtering all were removed except for Ca which was found throughout the oxide.
It is of a major interest to understand, on a time-scale from immediate responses to years, how material properties influence cellular activity in the interface and vice versa since rejection, excessive scar formation/encapsulation by fibrous tissue and restitution of original tissue may largely influence the performance of the implant. In soft tissues a fibrous capsule is formed around the implant (phenomenon of walling off the material from the biological environment) (Thomsen and Ericson, 1991). In bone, encapsulation of the implant by fibrous tissue may occur but is not obligatory and instead mineralized bone can establish direct contact with the implant, a process called osseointegration (Br{dot over (a)}nemark et al., 1969). Although the work on cell-material interactions has been intensified during recent years, the mechanisms by which material properties influence biological reactions are still not clear. Studies in vitro.
The attachment of tissues to implants in vivo is a complex matter because in most cases there are different types of tissues involved which may behave differently at different surfaces. The response of cells to variations in culture substrate topography varies for different cell types like macrophages (Rich and Harris, 1981; Salthouse, 1984), fibroblasts (van der Valk et al., 1983), periodontal cells (Cochran et al., 1994), epithelial cells (Chehroudi et al., 1989; Chehroudi et al., 1990), osteoblasts (Bowers et al., 1992; Martin et al., 1995) and chondrocytes (Schwartz et al., 1996).
Rich and Harris (1981) showed that macrophages accumulated preferentially on less hydrophilic as well as on roughened substrata. Murray et al (1989) showed that when macrophages adhered to hydrophilic surfaces PGE2 release and bone resorption was stimulated compared with hydrophobic surfaces. In addition, the rough surfaces was found to stimulate bone resorption to a greater extent than smooth surfaces. Although the roughness and the surface energy of the different surfaces were not quantitated this indicates that the interactions between macrophages and implant surfaces cause a release of factors which is higher than if cells are in suspension. Studies on human monocyte interactions with titanium surfaces have shown that the interleukin-1 release by the cells is modulated by protein adsorption and the presence of material particles (Gretzer et al., 1996).
Interestingly, different results have been obtained with fibroblasts. Human fibroblasts attached better to smooth than to rough titanium surfaces, (polished with 1 xcexcm diamond paste versus the rougher; prepared with 240 or 600 grit silicon carbide metallographic papers) (Keller et al., 1989). Spreading of fibroblasts was found to depend on the polar surface free energy (van der Valk et al., 1983) since at least on various polymer surfaces, low cell spreading was found on low polar parts. Sukenik et al (1990) modified titanium surfaces with different covalently attached self-assembled monolayers (four different chemical endgroups; CH3; Cxe2x95x90C; Br; Diol). The neuroblastoma cell attachment to the different surfaces was comparable but cell spreading was least pronounced on the most hydrophobic surface (CH3 and Cxe2x95x90C)
Osteoblasts are sensitive to subtle differences in surface roughness and surface chemistry and respond to altered surface chemistry by altering proliferation, extracellular matrix synthesis, and differentiation (Boyan et al., 1995). Osteoblasts exhibited different phenotypes when cultured on rutile or amorphous TiO2 surfaces, but with the same oxide thickness and degree of roughness. Differences were therefore suggested to be attributed to crystallinity alone (Boyan et al., 1995).
Osteoblasts have an initial greater attachment to rough, sandblasted titanium surfaces with irregular morphology but average roughness (Ra) parameters did not predict cell attachment and spreading in vitro (Bowers et al., 1992).
Proliferation and differentiation parameters in osteoblast-like cells were modified by growing cells on titanium discs with an increased roughness (15-18 xcexcm) (Martin et al., 1995). Interestingly, cells at different stages of differentiation responded differently to the same surface (Boyan et al., 1995; Schwartz et al., 1996).
A basis for most studies in vitro on the role of surface properties for cell function is the adhesion of cells to the surface of the culture dish. The resulting interactions between the cell and the surface, with or without adsorbed molecules, is therefore a fundamental and obvious part of the experimental set-up. In this context it may also be argued that the properties of the material surface as stimulating or inhibiting factors on cells could be over-emphasized in relation to other potential and maybe equally important modulating factors present in the vicinity of cells and surfaces in the complex biological situation in vivo.
Studies on titanium implants in bone (Sennerby et al., 1993a; Sennerby et al., 1993b), indicated that osteoblasts did not adhere to the implant surface and that formation of bone was not initiated at the surface. This observation suggests that the studies on the interaction between osteoblasts and titanium surfaces in vitro is of minor relevance. Nevertheless, studies in vitro, where various aspects of the complex in vivo situation can be studied in detail may be of great value but this requires that the conditions in vivo are considered when the in vitro system is designed.
On the basis of the published in vitro studies it may be concluded that the surface roughness appears to influence the cell proliferation albeit differently depending on the degree of cell maturation. Differences in surface properties may influence the cell attachment and proliferation although the mechanism is not clear. It is also evident that different cell types are differently influenced by the surface properties. However, so far there are few studies on the effect of modified titanium surfaces on cellular behaviour. A review of the literature on the in vivo response to titanium implants is therefore appropriate.
In general, histology, histochemistry and immunohistochemical techniques have been used for the evaluation of soft tissue reactions. Due to technical difficulties to obtain thin sections of an intact metal-tissue interface the ultrastructure of the interface tissue has been difficult to study (Ericson and Thomsen, 1995). However, for metals in soft tissues, an electropolishing method by which the bulk metal, but not the thin surface oxide layer, is removed (Bjursten, 1990) have made such studies possible.
The macrophage plays a pivotal role during healing of soft tissue around implants. The soft tissue response to titanium implants in rats is described by Thomsen and Ericson in (Br{dot over (a)}nemark et al., 1995). A fluid space, containing cells and proteins was present during the early phase (1-2 weeks) after introduction of a titanium implant in soft tissues (Johansson et al., 1992; Rxc3x6stlund et al., 1990). The concentration of leukocytes and the proportion of PMN in the fluid space decreased between 1 and 7 d (Eriksson et al., 1994). After one week the majority of inflammatory cells in the fluid space, predominantly monocytes and macrophages, were attached to the fibrin matrix at the border between the fluid space and the reorganized tissue rather than to the implant surface. After six weeks the fluid space was largely absent and the macrophages had established contact with the implant surface (Johansson et al., 1992; Rxc3x6stlund et al., 1990). Macrophages constituted the most common cell type at the titanium surface, and exhibited different phenotypes, as judged by their ultrastructure (Johansson et al., 1992). Immunohistochemical observations (Rosengren et al., 1993) show that the fluid space around a titanium implant one week after implantation contained albumin, complement factor C3c, immunoglobulins, fibrinogen and fibronectin. Albumin and C3c were distributed in the fluid space and throughout the tissue interstitium during the first week. Fibrinogen and fibronectin co-localized preferentially at the border between the fluid space and the tissue, thus forming a provisional matrix to which macrophages and fibroblasts adhered.
After 6 and 12 weeks, fibrinogen was not detected in the surrounding tissue whereas strands of fibronectin was found in the surrounding capsule (Rosengren et al., 1996). Collagen type I immunoreactivity, coinciding with the collagen bundles in the surrounding tissue, had a distribution similar to that of fibronectin, reaching close to the titanium surface, but always separated from it by one to several layers of macrophages after 12 weeks.
The general sequence of cellular migration and accumulation as well as the leakage of plasma into the tissue in the immediate vicinity of the implant surface has been observed after implantation of several different materials, including metals, in soft tissues (Thomsen and Ericson, 1991). The tissue response around nitrogen-ion implanted titanium discs inserted in the rat abdominal wall of rats was not significantly different from that observed around pure titanium implants. However, after 6 weeks a predominance of macrophages and multinuclear giant cells was found around the nitrogen-ion implanted discs (Rxc3x6stlund, et al., 1990). A comparison of titanium and Ti6Al4V after 1, 6 and 12 weeks in the same rat abdominal wall model did not reveal any differences with regard to cell types and numbers in the interface (Johansson et al., 1992). Further, the authors did not find any difference in fibrous capsule width. Therin et al. (1991) showed similar results when comparing the capsule thickness for titanium, TiO2-coated titanium, Ti6Al4V, TiO2-coated Ti6Al4V, TiN-coated Ti6Al4V, Ti5Al2.5Fe and stainless steel (316 L).
In contrast to polymers (Chehroudi et al., 1989; Chehroudi et al., 1990) studies in soft tissues which have been focused on the biological effects of altered surface topography and roughness of metal implants are relatively few.
However, in an extensive light microscopical study on the effects of surface roughness variations of titanium and stainless steel, (Ungersbxc3x6ck et al., 1994) it was shown that smooth implants induced a thicker soft tissue capsule with an intervening fluid space. In contrast, blasted and anodized titanium plates with relatively high values of roughness parameters (Ra 0.75) were surrounded by a significantly thinner soft tissue layer without a continuous liquid space. On the basis of these results it is difficult to conclude that there exists a simple relationship between increased surface roughness and capsule thickness. For instance, Al2O3-blasted titanium plates with an even greater surface roughness (Ra 1.5) had a capsule thickness which was similar to that around blasted, anodized titanium samples. Further, tumbled titanium plates (Ra 0.15) had a capsule thickness which was similar to tumbled and anodized smooth titanium (Ra 0.33). The roughness was measured with a profilometer and the elemental composition of implant surfaces was not reported. It is therefore possible that the surface chemical composition and/or roughness on the submicrometer level, differed between the samples. Studies on the effects of various surface topographies (smooth vs. various microtextures between 1 and 10 xcexcm) of titanium discs implanted in soft tissues of rabbits showed that collagen type III immunoreactivity was detected in the fibrous capsule around several materials, but that collagen type I was positively stained only in capsules around titanium (von Recum et al., 1993).
In general, the experimental studies in soft tissues indicate that metals become surrounded by a fibrous capsule with macrophages located closest to the surface, thus separating fibroblasts from the surface. So far there are few available morphological data on the interface structure around titanium surface modifications. It is still an open question how the material surface properties influence protein adsorption during in vivo conditions and how the surface properties influence the cells close to the surface. Moreover, it is not understood how the composition and structure of the surrounding fibrous capsule is influenced by the material surface-macrophage interactions. It is likely that several additional factors must be considered, including leaching of metal ions, loading conditions and micromovements between the implant surface and tissue.
The response of bone to injury is regeneration followed by remodelling of the newly formed bone in the direction of stresses. Analogously, when an implant is inserted in bone, a similar cascade of events is expected to occur including the recruitment of mesenchymal cells to the wound site, their differentiation into osteoblasts, synthesis of osteoid, and calcification of the extracellular matrix. The mesenchymal progenitor cells are pluripotent and able to differentiate into osteoblasts, chondrocytes, muscle cells and fat cells (Caplan and Boyan, 1994). The pathway of differentiation of the mesenchymal cells as well as regeneration of bone around an implant is most likely dependent on a combination of factors including the degree of trauma, local and systemic factors as well as implant properties and stability.
In the following a short summary of previous work on the interaction between metal implants and bone will be given. The performance of non-metal implants is reviewed elsewhere (de Groot et al., 1994).
Studies comparing the performance of, different implants of metals including Vitallium(copyright), niobium, titanium, titanium alloy, stainless steel (Johansson et al., 1991), and zirconium (Albrektsson et al., 1985; Johansson et al., 1994) in bone, did not reveal any major qualitative differences. The threaded titanium implants were in general found to be in contact with more mineralized bone than the other types of metal. The mechanisms for this is not clear nor is it understood why the properties of titanium are advantageous for biological applications compared with other metals, including those nearby in the periodic system. The good biological performance of titanium has been attributed to the titanium oxide layer covering the surface, but no compelling evidence for this view has been presented.
Several studies have indicated that an increased roughness of implant surfaces (within a certain range) enhance the biomechanical performance of implants. However, the bone response seldom show differences although some studies indicate an increased bone-implant contact with increased surface roughness (Buser et al., 1991; Goldberg et al., 1995; Gotfredsen et al., 1995). Most studies did not reveal such a correlation (Carlsson et al., 1988; Gotfredsen et al., 1992; Thomas and Cook, 1985; Thomas et al., 1985; Thomas et al., 1987; Wennerberg 1996; Wilke et al., 1990; Wong et al., 1995). Br{dot over (a)}nemark (1996) made a correlation between morphological parameters of osseointegration of threaded titanium implants and different biomechanical tests and found that pull-out tests mainly reflects the mechanics of the surrounding bone while removal torque tests reflects the shearing forces leading to plastic deformation of the bone-implant interface. Possibly biomechanical tests performed on implants with a rough surface (micrometer level), inserted in bone mainly reflect the bone-material mechanical interaction (interlocking) although it cannot be excluded that differences in the structure of the interface not resolved by light microscopy are of importance.
Using an animal model similar to that used in the present study Sennerby et al (1993b), studied the bone response 3-180 days after insertion of screw-shaped titanium implants. At 3 days mesenchymal cells were migrating into the injury area around the implants. The implant surface was temporarily covered by multinuclear giant cells which disappeared with time and when bone-titanium contact increased. Newly formed bone extended from the endosteal surface towards the implant and was also formed as islands within the implant threads.
With time the two types of newly formed bone fused. The threads originally protruding into the marrow cavity were gradually filled with bone which matured by remodelling. Formation of new bone directly at the titanium surface was not observed at any time interval.
Only a limited number of studies of the ultrastructure of the bone-metal interface tissue are available. This may reflect the fact that the preparation of the interface tissue for analysis by transmission electron microscopy TEM is technically demanding, especially when the decalcification step is omitted.
Albrektsson et al (1982) introduced polycarbonate plugs coated with a thin layer of evaporated metal as a model for metal implants. The plugs were implanted in the rabbit tibia. TEM on partially decalcified specimens showed the presence (after 3 months) of collagen bundles close to titanium implant but the last 100-500 nm closest to the implant consisted of randomly arranged filaments. A 20-40 nm thick layer of partially calcified amorphous substance, suggested to consist of proteoglycans was found in contact with the implant surface. A gradient of decreasing mineralization towards the implant surface was also described. In contrast, a larger number of macrophages and osteocytes were found at gold-coated plugs. In more recent studies based on the plastic plug technique, other metal coatings including zirconium has been compared with titanium (Albrektsson and Hansson 1986; Albrektsson et al., 1985).
Linder et al (1989), studied the interface morphology of plugs of titanium. Ultrastructural observations in rabbit cortical bone (11 months observation period) adjacent to titanium, Tivanium(copyright), Vitallium(copyright), and stainless steel revealed an unpredictable variation in interface ultrastructure within 500-1000 nm of all metal surfaces. Three main types of interface structure were found; a) More or less regularly arranged fibrils of collagen, with the longitudinal cross-banding of 68 nm typical of type-I collagen, approaching the metal surface to within 50 nm: b) Type-I collagen fibrils separated from the implant by a zone of indistinct structures, but with some filamentous material, most often about 500 nm in thickness, but sometimes up to 1000 nm; c) Type-I collagen fibrils separated from the implant by a 500-600 nm zone of thin filamentous structures, clearly more dense than in b. There was no structural feature that was specific for a particular material (Linder et al., 1989).
Sennerby et al (1992) examined the interface morphology of titanium implants inserted into the rabbit tibia for 12 months and found mineralized bone to be present very close to the implant surface without any apparent decreasing gradient of the concentration of bone mineral towards the implant surface. A thin layer of amorphous non-mineralized material (100-200 nm wide) was present peripheral to the mineralized bone. In addition, visible when mineralization was low, an about 100 nm wide electron dense lamina limitans was found to form the border between mineralized bone and the amorphous layer. This lamina limitans were often seen in direct continuity with lamina limitans bordering osteocyte canaliculi or separating bone of different mineralization grades.
Steflik studied the interface morphology at various types of implants in the dog mandible using TEM and high voltage TEM and found an about 50 nm wide electron dense deposit at the implant surface (Steflik et al., 1992a; Steflik et al., 1992b). No difference was seen between loaded and unloaded implants (Steflik et al., 1993).Nanci et al (1994) studied the tissue response to titanium implants inserted for 1 day to 5 months in tibia and femur of rats. The morphology of the interface tissue varied. Most often the interface between bone and the titanium implant consisted of a thin, electron-dense layer. This interfacial layer was found both adjacent to mineralized bone and unmineralized collagen. With immunocytochemical techniques, the electron-dense layer described as lamina limitans was shown to be immunoreactive for osteopontin. The cement lines in the surrounding bone often in continuity with the lamina limitans at the implant surface, showed a similar immunoreactivity for osteopontin. Osteocalcin, fibronectin, and albumin showed no preferential accumulation at the interface. In a recent study McKee and Nanci, (1996) are suggesting that osteopontin functions as a mediator of cell-matrix and matrix-matrix/mineral adhesion during the formation, turnover and repair of mineralized tissue. A review of the literature on the soft tissue response to titanium implants is important since a penetration through skin and mucous membranes is necessary to allow the attachment of external prosthetic appliances (e.g. teeth and epistheses). Interest has been focused on the prerequisites for an adequate adaptation of the soft tissue to the penetrating element. Empirically it has been found that a careful surgical technique with minimal motion at the interface by a tight adherence of the soft tissues to the underlying bone may provide adequate conditions for clinical percutaneous and permucosal implants/anchorage units.
In studies on the relationship between the titanium surface and epithelium and connective tissues the majority of observations in humans have been made in specimens from the oral cavity (Sanz et al., 1991; Seymour et al., 1989; Tonetti et al., 1993) and from the craniofacial region (bone conductive hearing aids) reviewed in (Holgers 1994). In a light microscopic and ultrastructural study of oral implants (Sanz et al., 1991) the inflammatory infiltrates were scarce in the non-infected peri-implant tissue. However, when gingivitis was observed, the inflammatory infiltrates were larger, dominated by mononuclear cells and plasma cells. (Seymour et al., 1989) characterized the mucosa around Br{dot over (a)}nemark osseointegrated titanium implants. The samples were obtained from healthy mucosa or with clinical signs of inflammation (gingivitis). The authors reported the presence of inflammation in both situations (healthy gingiva or gingivitis) but found larger inflammatory infiltrates and higher cell numbers when clinical signs of gingivitis were present. The authors concluded that the mucosal reaction was a stable and well controlled response. Similar findings were reported around clinically functioning bone-anchored percutaneous implants (Holgers, 1994), suggesting that an immunological compensation for the loss of barrier function is present at implants with clinically irritated skin. The relationship between epithelial cells and the surface of implants as well as the common observations of epithelial downgrowth have been suggested to play an important role for the function of implants, both in oral and percutaneous applications. In contrast to observations for dental implants (Listgarten and Lai, 1975; Schroeder et al., 1981), no close contact between the epithelium/collagenous tissue and the surface of percutaneous titanium implants were seen (Holgers et al., 1995).
In conclusion, these observations indicate that machined titanium implants in soft tissues of humans are surrounded by inflammatory cells which appear to provide a protective barrier which may compensate for a non-optimal epithelial barrier.
Analysis of a retrieved osseointegrated clinical titanium implant (3 months) (Lausmaa J. 1988) revealed an increased oxide thickness (by factor 2-3) compared with an unimplanted sample. Similar in vivo oxide growth have been reported earlier. By the use of Auger electron spectroscopy, McQueen et al. (1982) observed that after 6 years in human jaw bone, the original 50 xc3x85 thick oxide layer on titanium implant surfaces had increased to a 2000 xc3x85 thick oxide layer.
Sundgren et al (1986) investigated the interface of bone-titanium and bone-stainless steel in humans and found that both the thickness and the nature of the oxide layers on the implant had changed during the time of implantation. Depending on the location, the thickness remained unaffected (cortical bone) or increased with 3-4 times (bone marrow). In both cases, Ca and P were incorporated in the oxides. For titanium implants the oxidation process occurred over a longer time period (several years).
In a light microscopical study by Sennerby et al. (1991), seven clinically stable (1-16 years) osseointegrated dental implants, were analyzed morphometrically. The major part of the implants were in contact with mineralized bone (56-85%), irrespective of observation period. Carlsson et al (1994) evaluated the tissue around implants with different roughness inserted experimentally in arthritic knees. Blasted titanium and hydroxyapatite-coated implants were in contact with bone whereas smooth titanium implants often were surrounded by fibrous tissue .
Sennerby et al (1991), examined the structure of the interface around seven clinically stable dental implants (1-16 years) by morphometry. In areas with mineralized bone close to the titanium surface, a non-mineralized amorphous layer was observed. An electron dense lamina limitans-like line was observed between the mineralized bone and the 100-400 nm wide amorphous zone.
Ultrastructural observations were made on the metal-bone of interface of implants inserted in the tibia of patients with arthrosis and rheumatoid arthritis (7-20 months) (Serre et al., 1994) The implants were all screw-shaped pure titanium implants and they were all xe2x80x9cosseointegratedxe2x80x9d. No difference between the ultrastructure of the interface between normal bone and implants compared with the interface of arthrotic and arthritic bone was observed. The heterogeneity of the interface was also confirmed in this study although the 100-400 nm wide amorphous zone reported by Sennerby et al (1991), was not found.
In an ultrastructural study of the interface of a plasma-sprayed titanium dental implant inserted in man (ITI), (Hemmerlxc3xa9 and Voegel, 1996), two different interfacial structures were noticed. Both bone crystals directly apposed on the implant surface and a granular electron-dense substance interposed between the plasma-sprayed coating and the bone were observed. Rohrer et al (1995) examined non-decalcified histologic sections from 12 osseointegrated titanium plasma spray-coated (TPS) and TPS-treated with hydroxyapatite implants (IMTEC) from one patient. All implants were successful and stable after 1 year when the samples were retrieved. Both implant types were used with the same success and no morphological differences were observed between the two implant types.